We have undertaken to study the metabolic and clinical significance of the cobalamin (Cbl) analogues recently discovered in animal serums and tissues. In a series of experiments, we have found that calf kidney contains a Cbl analog that stimulates the activity of methyltransferase from prokaryotic and eukaryotic sources. Cbls extracted in KCN from fresh kidneys were measured by radioisotope dilution assay with R-protein binder. Kidney extract was applied to a reverse affinity column (made of R-protein coupled to cyanogen bromide-activated Sepharose). Cbl was eluted with phenol, and passed, through a Dowex-50 (NH4 ion) column. Cyanocobalamin (CN-Cbl) was eluted with water. The column was then washed successively with buffers from pH 3.5 to 6.5. A sharp minor Cbl peak, representing 0.5 percent of total Cbl, appeared at pH 4.1. It was not hydroxo-, adenosyl-, or methylcobalamine and was relatively less active than CN-Cbl on radioassay with IF binder and microbiological assay with Lactobacillus leichmannii. This material when tested as cofactor for methyltransferase activity in extracts of rat liver and E. coli, was a more potent methyltransferase cofactor than CN-Cbl. Studies are now in progress on the activity of this and other analogues with cobalamin adenosylating enzyme, methylmalonyl CoA mutase, and other cobalamin-dependent enzymes.